Article The Gut Microbiota of Naturally Occurring and Laboratory Aquaculture Lytechinus variegatus Revealed Differences in the Community Composition, Taxonomic Co-Occurrence, and Predicted Functional Attributes George B. H. Green 1,†, Joseph A. Hakim 1,†,‡, Jiung-Wen Chen 1 , Hyunmin Koo 1,§ , Casey D. Morrow 2, Stephen A. Watts 1 and Asim K. Bej 1,* 1 Department of Biology, The University of Alabama at Birmingham, 1300 University Blvd., Birmingham, AL 35294, USA; [email protected] (G.B.H.G.); [email protected] (J.A.H.); [email protected] (J.-W.C.); [email protected] (H.K.); [email protected] (S.A.W.) 2 Department of Cell, Developmental and Integrative Biology, The University of Alabama at Birmingham, 1918 University Blvd., Birmingham, AL 35294, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-205-934-9857 † Equal contribution by these authors presented alphabetically. ‡ Current address: School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA. § Current address: Department of Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Citation: Green, G.B.H.; Hakim, J.A.; Abstract: Sea urchins, in many instances, are collected from the wild, maintained in the laboratory Chen, J.-W.; Koo, H.; Morrow, C.D.; aquaculture environment, and used as model animals for various scientific investigations. It has Watts, S.A.; Bej, A.K. The Gut been increasingly evident that diet-driven dysbiosis of the gut microbiome could affect animal Microbiota of Naturally Occurring health and physiology, thereby impacting the outcome of the scientific studies. In this study, we and Laboratory Aquaculture compared the gut microbiome between naturally occurring (ENV) and formulated diet-fed laboratory Lytechinus variegatus Revealed aquaculture (LAB) sea urchin Lytechinus variegatus by amplicon sequencing of the V4 region of the Differences in the Community 16S rRNA gene and bioinformatics tools. Overall, the ENV gut digesta had higher taxa richness with Composition, Taxonomic an abundance of Propionigenium, Photobacterium, Roseimarinus, and Flavobacteriales. In contrast, the Co-Occurrence, and Predicted Functional Attributes. Appl. Microbiol. LAB group revealed fewer taxa richness, but noticeable abundances of Arcobacter, Agarivorans, and 2021, 1, 201–224. https://doi.org/ Shewanella. However, Campylobacteraceae, primarily represented by Arcobacter spp., was commonly 10.3390/applmicrobiol1020016 associated with the gut tissues of both ENV and LAB groups whereas the gut digesta had taxa from Gammaproteobacteria, particularly Vibrio spp. Similarly, the co-occurrence network displayed Academic Editor: Bong-Soo Kim taxonomic organizations interconnected by Arcobacter and Vibrio as being the key taxa in gut tissues and gut digesta, respectively. Predicted functional analysis of the gut tissues microbiota of both Received: 20 May 2021 ENV and LAB groups showed a higher trend in energy-related metabolisms, whereas amino acids, Accepted: 1 July 2021 carbohydrate, and lipid metabolisms heightened in the gut digesta. This study provides an outlook of Published: 14 July 2021 the laboratory-formulated diet-fed aquaculture L. variegatus gut microbiome and predicted metabolic profile as compared to the naturally occurring animals, which should be taken into consideration for Publisher’s Note: MDPI stays neutral consistency, reproducibility, and translatability of scientific studies. with regard to jurisdictional claims in published maps and institutional affil- Keywords: metagenome; CoNet; PICRUSt2; QIIME2; echinoderm; deuterostome; 16S rRNA iations. 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Organisms from all domains of life cohabit in the nearshore marine ecosystems world- This article is an open access article wide, where they thrive by modulating their community structure with the fluctuating distributed under the terms and biotic and abiotic factors, food sources, and other perturbances, such as natural calamities conditions of the Creative Commons and human activities [1–3]. The resiliency of these ecosystems in such changing envi- Attribution (CC BY) license (https:// ronments is often contingent upon the composition and metabolic activities of microbial creativecommons.org/licenses/by/ communities that help sustain some of the crucial trophic functions [2]. However, often- 4.0/). times organisms from their natural habitat are collected and transferred to laboratories and Appl. Microbiol. 2021, 1, 201–224. https://doi.org/10.3390/applmicrobiol1020016 https://www.mdpi.com/journal/applmicrobiol Appl. Microbiol. 2021, 1 202 used as model animals for various scientific studies. These organisms are maintained in the laboratory aquaculture environment where they are fed formulated feed with balanced nutrients. Studies have shown that such controlled laboratory conditions and feeding lab-standardized or commercially available diet reshape gut microbiota from their natural habitat in a variety of laboratory model animals such as Mus musculus [4], Danio rerio [5], Caenorhabditis elegans [6,7], and Drosophila [8]. The pivotal role of diet and gut microbiome in maintaining host metabolisms, health, and disease has been a well-established fact now [9–11]. Dysbiosis of the gut microbiota as a result of improper selection of diet thereby affecting the homeostasis of laboratory animal models has been a growing concern in the outcome and interpretation of various scientific studies [12–15]. Oftentimes, the sea urchins, including the green sea urchins, Lytechinus variegatus (order Temnopleuroida, family Toxopneustidae), are collected from their natural habitats, acclimated, and maintained in the laboratory aquaculture, fed a laboratory-standardized formulated diet, and used for basic and applied research [16,17]. L. variegatus inhabit the area along the U.S. South-Eastern Coastal waters and into the Gulf of Mexico; although considered omnivorous, they primarily graze upon seagrass, algae, and decomposed mate- rials in their natural habitats [18–21]. L. variegatus represents a characteristic deuterostome gut system [22], in which the ingested food along with the natural microbial communities from their habitat is encapsulated in the pharynx by a thick mucus layer known as gut digesta. The microbiota in the gut digesta remains separated from that of the gut tissue during their passage through the gut lumen until egested [23–26]. The culture-dependent studies of urchins from their natural habitat have shown representative bacteria from the gut capable of alginolytic activities [27]. In addition, gelatin, protein, and amino acid assimilation by the gut microbiota have also been elaborated in urchins [28–30]. However, the laboratory-maintained urchins are normally fed a diet consisting of combinations of naturally occurring algal and seagrass sources [16,31] or a formulated feed optimized for their nutritional requirements and health [32]. These conditions contrast the food in their natural habitat and the diverse marine microorganisms associated with it [33–40]. Consequently, the laboratory-standardized formulated diet could potentially reshape the gut microbiota and metabolic role in laboratory aquaculture L. variegatus. In this study, we elucidate a comparative outlook of the gut microbial community compositions along with the taxonomic co-occurrence network and predicted metabolic profiles in naturally occur- ring and formulated diet-fed laboratory aquaculture L. variegatus using 16S rRNA-targeted metagenomics approach and bioinformatics tools. 2. Materials and Methods 2.1. Sample Description and High-Throughput Sequencing Adult L. variegatus were collected from Saint Joseph Bay Aquatic Preserve, Florida (29.80◦ N 85.36◦ W), and transported to the University of Alabama at Birmingham (UAB) and held for six months in a recirculating saltwater tank system before tissue collection and processed for the microbiome analysis (LAB group; n = 3), as described elsewhere [41]. The LAB urchins were fed ad libitum once every 24–48 h with a formulated feed consisting of 6% lipid, 28% protein, and 36% carbohydrate relative percentages [32], and the aquaria conditions were maintained at 22 ± 2 ◦C with a pH of 8.2 ± 0.2 and salinity of 32 ± 1 parts per thousand (ppt.). Similarly, adult L. variegatus (n = 3) from the same location (29.80◦ N 85.36◦ W) with 1 m2 area were collected and transported to UAB (ENV group), and tissues were retrieved and processed for microbiome analysis within 7 ± 1 h after collection. The conditions of the water were recorded as follows: 20 ± 2 ◦C with a pH of 7.8 ± 0.2 and salinity of 28 ± 1 ppt. during sample collection [42]. The gut tissues and the digesta were collected from urchins using the procedures described elsewhere [24]. Briefly, a radial incision was made around the Aristotle’s Lantern mastication apparatus of the urchins using sterile instruments. The pharynx enclosed by the lantern was separated from the digestive tract, collected intact, without tearing, and rinsed with sterile phosphate-buffered saline water (1× PBS, pH 7.4) (Fisher Scientific, Hampton, NH, USA). The gut tissues were Appl. Microbiol. 2021, 1 203 then carefully opened by incision and the digesta were separated by gently rinsing with sterile 1× PBS (pH 7.4). The gut tissues were examined under a dissecting scope to make sure no residual digesta associated with the tissues before subjected to DNA purification. Similarly, the gut digesta were gently rinsed multiple times in sterile 1× PBS (pH 7.4) to remove